CELL-SURFACE
ANTIGENS
RECOMBINANT
MINK
MURINE
ASSOCIATED
WITH
CELL FOCUS-INDUCING
LEUKEMIA
VIRUSES
BY MILES W. CLOYD,* JANET W. HARTLEY, AND WALLACE P. ROWE From the Laboratory of Viral Diseases, National Institute of Aller~ and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 The endogenous retroviruses of mice, which exist as normal, inherited components of the mouse genome, are generally classified on the basis of their species host range as ecotropic (capable of infecting only mouse or rat cells), xenotropic (capable of infecting only heterologous species cells), or amphotropic (wild mouse viruses capable of infecting both mouse and heterologous species cells). Recently, a new class of murine leukemia virus (MuLV) 1 was described which has properties of both xenotropic and ecotropic viruses and which induces morphological changes in the mink lung cell line A T C C No. CCL-64 (1). Abundant evidence indicates that these viruses, designated mink cell focus-inducing (MCF) viruses, are env gene recombinants between ecotropic and xenotropic MuLVs (1-4). The emergence of MCF viruses from lymphoid tissue during the preleukemic or leukemic state may indicate that they are involved in some way in the pathogenesis of murine leukemia (1). To obtain more understanding about the natural history of M C F viruses, we have attempted to develop MCF-specific serological probes which could be applied in identification, classification, and characterization of in vivo expression of MCF viruses. In this report we describe the specificity of these reagents and the corresponding cellsurface antigens induced in cell cultures infected with M C F viruses. Reagent rabbit antisera prepared against A K R MCF-infected rabbit cells were able to distinguish major antigenic determinants specific for ecotropic, xenotropic, and three subclasses of MCF viruses. Materials and Methods Viruses and Cells. Naturally occurring MCF viruses were isolated from mice by plating mitomycin C-treated lymphoid cells as infectious centers onto CCL-64 mink cells as described previously (1). The viruses were adapted to cell-free passage in SC-1 (5) and/or mink lung cell cultures, and were carried through two cycles of limiting dilution purification. Table I lists the viruses and cells used in this study. Tests with Moloney MCF were done chiefly with a strain isolated by Dr. Akinori Ishimoto of this laboratory; however, the prototype recombinant virus from Moloney stocks, HIX (4), kindly supplied by Dr. Peter Fischinger, National Institutes of Health, was also used and was found to be serologically identical to this Moloney MCF isolate. HIX and Moloney MCF are therefore used interchangeably throughout this report. Most of * Recipient of a Fellowship from the Damon Runyon-Walter Winchell Cancer Fund, N. Y. 1 Abbreviations used in this paper: MuLV, murine leukemia virus; MCF, mink cell focus-inducing; env,
envelope; IUdR, 5-iododeoxyuridine; MiLV, mink leukemia virus; ecotr., ecotropic; Xtr., xenotropic; Amtr., amphotropic.
702
THE J O U R N A L OF E X P E R I M E N T A L MEDICINE • V O L U M E 149, 1979
703
CLOYD, HARTLEY, AND ROWE TABLE I Description of Murine Leukemia Viruses Used Class Ecotropic
Strain
Thymus of 2-mo-old AKR Mouse
AKRL1
Spleen, thymus, and nodes from spontaneous AKR leukemia. Tail extract of young Akv-I congenie mouse. NB-tropic virus isolated from mouse-passaged Moloney stock. NB-tropic virus isolated from BALB/c passed stock. BALB/C embryo cells treated with IUdR. Thymus of 2-mo-old AKR mouse. Thymus of 6-mo-old A K R mouse NZB-Q cell line treated with IUdR. Isolated by Todaro et al. (6) from an NIH Swiss mouse. IUdR-treated embryo cells of wild mouse from Lake Casitas (Calif.) area. Embryo cells of wild mouse from Lake Casitas (Calif.) (7, 8). Embryo cells of wild mouse from LaPuente (Calif.) area. Thymus of normal 6-mo-old A K R / J mouse (1). Thymus of 3-too-old A K R / J mouse that had received thymus graft from 6-mo-old A K R / J 1 mo previously. Spontaneous lymphoma of NFS mouse partially congenic for Akv-2. Spontaneous reticulum cell sarcoma of NFS mouse partially congenic for Akv-2. Spontaneous lymphoma of NFS mouse partially congenic for Akv-1. Spontaneous reticulum cell sarcoma of NFS mouse partially congenlc for Akv-l. Spontaneous reticulum cell sarcoma of NFS mouse partially congenic for a C58/Lw virus-inducing locus. Spontaneous Hodgkin's-type reticulum cell sarcoma of NFS mouse partially congenic for a C58/Lw virus-induclng locus. Spontaneous lymphoma of NFS mouse partially congenie for C3H/Fg virus-inducing locus Fgv-l. Pristane-induced plasmacytoma ofa BALB/c mouse. Moloney stock passed in BALB/c mice (A. Ishimoto, unpublished observations). IC-Strain of Moloney MuLV (4). Friend stock passed in BALB/c mice (A. Ishimoto, unpublished observations).
RS-18 MIT Moloney
Xenotropic
Friend BALB-IU-1 AKR6 AKR40 NZB-IU-3 AT124 Cas.E 1
Amphotropic
4070A 1504A
MCF
Source
AKR2a
AKR-247 AKR-13
Akv-2-C34* Akv-2-C26 Akv- l-C36 Akv- 1-C44 C58-C301-45
C58-C298-48
Fg-C207-647 CB208 Mol. MCF HIX Friend MCF
Grown In* NIH/3T3 FRE, SC-1 NIH/3T3 SC- 1 NIH/3T3 FRE, SC-1 Mink Mink Mink Mink Mink Mink Mink Mink SC-I Mink Mink
Mink Mink Mink Mink Mink
Mink
Mink Mink Mink Mink Mink
* Cells include NIH/3T3; Fisher rat embryo (FRE C12, from Dr. Edward Scolnick, NIH); SC-1 (a cloned line of wild mouse embryo cells); and mink lung cells (CCL64). :]: The C in the virus designation refers to its origin from a congenic mouse.
704
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
the MCF isolates were N-tropic. Exceptions include an isolate (CB208) from a BALB/c plasmacytoma, which was B-tropic; and Moloney, HIX, and Friend MCF viruses, which were NB-tropie. Mink cells replicating an endogenous mink C-type virus (MiLV) were kindly supplied by Dr. Raymond Gilden, Frederick Cancer Research Center, Ft. Detrick, Frederick, Md. All cells were grown in the Dulbecco-Vogt modification of Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal calf serum, 2mM glutamine, 250 U / m l penicillin, and 250 ~g/ml streptomycin. Antisera and SerologicalAssay. MCF antisera were raised in New Zealand white rabbits by a single intravenous injection of 2 × 10v MCF-infected SIRC cells as described elsewhere (9); SIRC is a tissue culture line of rabbit corneal cells (10). Anti-AKR-247 MCF serum 827 was obtained from a rabbit 3 wk after injection, precipitated with sodium sulfate, dialysed, and reconstituted at a four-fold concentration. It was kindly supplied by Dr. Thomas Chused, National Institutes of Health. Anti-AKR-247 MCF serum R25461 and anti-AKR-13 MCF serum R25415 were from pooled weekly bleedings taken 2-5 wk after injection. Control sera included pre-immune sera and an anti-SIRC cell serum, prepared in the manner described above using uninfected SIRC cells. An indirect live-cell immunofluorescenee assay described previously (11) was used, with slight modification, to score for antibody activity. Briefly, 1 × 105 freshly suspended cells were mixed with 50 #1 (total vol, 60/~1) of diluted rabbit antiserum in wells of a microtiter plate for 20 min at room temperature, rinsed, and then incubated similarly with 50/~1 of appropriately diluted fluorescein-eonjugated goat anti-rabbit IgG (Meloy Laboratories Inc., Springfield, Va.). After rinsing, the cells were examined under epi-UV illumination at 300 times magnification, and the percentage of cells showing surface fluorescence determined. Absorptions. To determine antibody specificity, qualitative absorptions were performed. The experimental design was to absorb with one set of antigens (virus-infected cells) and then test the absorbed antiserum on a panel of target cells. The procedure, described in detail elsewhere (12), entailed mixing, in duplicate, 5 X 10v freshly trypsinized tissue culture cells with 0.5 ml of antiserum diluted about fourfold less than the 50% staining point for the chosen target cell. After overnight incubation at 4°C, the sera were centrifuged; the serum from one tube was frozen, and the serum from the other was mixed with another 5 × 10 7 cells for a second absorption. The one- and two-times absorbed antisera were then tested on each target cell; if the second absorption did not significantly reduce the antibody activity (as determined by percent cells stained) below that seen after one absorption, the antiserum was considered completely absorbed. If the activity was significantly decreased with the second absorption but some activity remained, then an additional absorption was performed. Residual staining activity for a target cell was therefore interpreted as demonstrating that an antigenic determinant detected on that target cell was not present on the absorbing cell. All the absorption data reported herein were obtained with doubly absorbed antisera; three absorptions were not required. Results
Activity of Anti-AKR-247 MCF Serum for Cells Infected with Various MuL Vs.
Three antisera p r e p a r e d against A K R - 2 4 7 M C F - i n f e c t e d S I R C cells (antiserum 827, R25461, a n d a h y p e r i m m u n e serum, 48, which was not studied further) were t i t r a t e d in the m i c r o - i m m u n o f l u o r e s c e n c e test against various virus-infected a n d control cells to ascertain the extent o f their a n t i b o d y activity. All were c o m p a r a b l y active against the various M u L V - i n f e c t e d cells; the activity of a representative serum (827) is s u m m a r i z e d in T a b l e II. U n i n f e c t e d mink, SC-1, or S I R C cells, a n d m i n k cells r e p l i c a t i n g an e n d o g e n o u s m i n k C - t y p e virus (13) were not significantly reactive with this a n t i s e r u m , i.e., the 50% s t a i n i n g titers were between 1:2 a n d 1:5. Cells infected with M o l o n e y or F r i e n d ecotropic viruses were slightly reactive (50% titers of 21:10), a n d cells infected with A K R - t y p e ecotropic viruses gave 50% serum titers o f 1:60 to 1:75. T h e a n t i s e r u m was also significantly active against cells infected with xenotropic a n d a m p h o t r o p i c
705
CLOYD, HARTLEY, AND ROWE TABLE II Anti-AKR-247 MCF Serum (827) Activity for Cells Infected with Various C-Type Viruses Control cells
Mink SC-I SIRC Mink infected with MiLV
50%* titer 2 5 5
EcotroF" (cell)
50%* titer
MIT Moloney (FRE) MIT Moloney (SC- 1) Friend (Mink)
10 12 12
AKR2a (NIH/3T3) AKR2a (FRE)
60 65
AKR2a (SO-l)
75
50%* titer
Amphotropic virus (cell)
50%* Titer
MCF virus (cell)
50%*
ATI24 (Mink) AKR40 (Mink) AKR6 (Mink)
20 30 3O
4070A(Mink) 1504A(SO-1)
20 18
I00 250 250
NZB-IU-3 (Mink) BALB-IU-1 (Mink)
50 7O
Friend (Mink) Mol.-HIX (Mink) C58-C301-45 (Mink) Akv-2.C26 (Mink) C58-C298-48 (Mink) Akv-2-C34 (Mink) A K R I 3 (Mink) Akv-I-C44 (Mink) Akv- I-C36 (Mink) Fg-C207-647 (Mink) CB208 (Mink) AKR-247 (Mink)
Xenotropic virus (ceil)
titer
250 250 300 3OO 340 340 340 4OO 64O
* Reciprocal of antiserum dilution that stains 50% of the cells.
MuLVs; the highest titer for xenotropic-infected cells was that for the BALB-IU-1 strain (50% titer 1:70). Lesser activities were found for NZB, AKR, and NIH Swiss xenotropic viruses and for wild mouse amphotropic MuLV-infected cells. The antiserum displayed the greatest activity for MCF MuLV-infected cells (50% titers ranging from 1:100 to 1:640), with cells infected with the homologous AKR-247 MCF virus giving the highest titer. Pre-immune and rabbit anti-SIRC cell control antisera were also tested on approximately one-half of the cells and were found not to be significantly reactive, giving 50% staining titers between 1:1 and 1:3 (data not shown). In neutralization tests, the MCF-247 antiserum displayed similar activities, having slight neutralization titers for ecotropic and xenotropic MuLVs (< 1:20) but significant activity for MCF viruses (1:40-1 : 160). From these studies, it was concluded that the anti-AKR-247 MCF serum was reacting specifically with MuLV-associated cell-surface antigens and that the antiserum most likely contained multiple MuLV specificities. Qualitative Absorptions to Determine Serological Specificity. The specificity of the antibody activity for the various MuLV-producing cell lines was analysed by absorptions. Antiserum 827 was absorbed with various MuLV-infected cells and then tested for activity on various virus-infected cells (Table III). Absorbing with uninfected mink, SIRC, or N I H / 3 T 3 cells did not appreciably reduce the antiserum activity (at a 1:10 dilution) for ecotropic, amphotropic, or xenotropic MuLV-infected cells or (at 1:801:100 dilutions) for MCF cells. These absorptions not only served as absorption controls, showing that nonspecific absorption was not involved, but also demonstrated that cellular, nonviral antigens were not being detected. Absorption with cells infected with AKR ecotropic MuLV removed only the antibody activity for the homologous target cells (AKR2a); the activities for cells infected with amphotropic, xenotropic, and three MCF M u L V strains were but slightly reduced. In addition to this AKR ecotropic MuLV isolate, two other ecotropic viruses from AKR (AKRL1 and RS-18) were tested for their absorption efficiency; the three strains absorbed identically and were completely cross reactive (data not shown). Absorptions with Moloney ecotropic virus-infected cells slightly diminished
706
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS TABLE III
Specificity of Anti-AKR-24 7 MCF Serum for MuL V-Infected Cells as Defined by Absorptions Cells used for absorption*
Virus type
None
Ecotr. Amtr. Xtr. Ecotro + Xtr MCF
Residual antiserum activity for cells* infected with these MuLVs AKR2a ecotropic
4070A amphotropic
Strain
BALBIU-1 xenotropic
Antise- AntiseAntiserum rum rum l:10 1:10 1:10 None Mink lung SIRC NIH/3T3 AKR2a Mol. 4070A BALB-IU-1 AKR2a + BALB-IU-1 Mol. + BALB-IU-1 Mol.-HIX
Akv-2-C26 AKR-247
Mol.HIX MCF
Akv-2C26 MCF
AKR247 MCF
Antise- Antise- Antiserum rum rum 1:80 l:100 1:100
91:]: 90 90 90 0 83 90 90 0
69 62 60 58 57 51 0 0 0
100 100 100 100 82 80 50 0 0
90 89 86 83 81 58 90 62 51
92 90 89 91 85 88 90 83 57
100 100 100 100 98 100 100 95 72
75 80 0 0
0 0 0 0
0 0 0 0
30 0 0 0
NT§ 54 0 0
84 79 76 0
* MCF, amphotropic, and xenotropic MuLVs were grown in mink lung cells (CCL 64). Ecotropic MuLVs were grown in NIH/3T3 cells. :~Percentage of cells fluorescing. § NT, not tested. the a n t i s e r u m activity for M o l o n e y - H I X M C F cells a n d did not significantly reduce a n y other activities, i n c l u d i n g that for A K R ecotropic M u L V . A b s o r b i n g with cells infected with wild mouse a m p h o t r o p i c virus e l i m i n a t e d the a n t i b o d y activity for the homologous target cells (4070A) a n d reduced by one-half the activity for BALB-IU-1 xenotropic M u L V cells, b u t it did not affect the activity for cells infected with A K R ecotropic or M C F viruses. Absorption with cells infected with B A L B / c xenotropic M u L V (BALB-IU-1) e l i m i n a t e d the a n t i b o d y activity for both a m p h o t r o p i c a n d xenotropic M u L V - i n f e c t e d cells, b u t only slightly reduced the activity for ecotropic- or M C F - i n f e c t e d cells. It thus appears that the a m p h o t r o p i c M u L V specificity detected with the 827 serum is reactive with a n a n t i g e n shared with xenotropic viruses. T h e a n t i s e r u m therefore c o n t a i n e d a specificity for ecotropic M u L V , not removed by absorption with xenotropic or a m p h o t r o p i c M u L V s , a n d a distinct specificity for x e n o t r o p i c - a m p h o t r o p i c M u L V s , not removed by absorption with ecotropic M u L V infected cells. T h e activity for M C F cells, not b e i n g absorbed by either amphotropic, xenotropic, or A K R ecotropic M u L V - i n f e c t e d cells, was clearly not one of these respective specificities. Also, as shown in T a b l e III a n d e x a m i n e d in more detail in a later section, this activity was not removed by absorptions with mixtures of ecotropic a n d xenotropic M u L V - i n f e c t e d cells. This indicated that a significant M C F - t y p e specificity was present in this antiserum.
CLOYD, HARTLEY, AND ROWE
707
Absorptions were also performed with the MCF MuLV-infected cells to determine if they expressed the ecotropic and xenotropic MuLV-specific determinants and to explore the relationships between the different MCF isolates. Ceils replicating MCF MuLVs of A K R and Akv-2 congenic origin were able to absorb completely both the A K R ecotropic and xenotropic type-specificities (Table III), showing that they do express both sets of determinants. Cells infected with Moloney-HIX MCF M u L V demonstrated a xenotropic component, but they were not cross-reactive with A K R ecotropic virus; this further demonstrates that there are significant differences between Moloney and A K R ecotropic components. Differential absorption of MCF activities by different MCF cells showed that at least three distinct MCF type-specific determinants could be discriminated. Moloney-HIX MCF was able to completely absorb the antibody activity for itself, but partial activity remained for two other MCF isolates. Cells replicating Akv-2-C26 MCF were able to completely absorb the antibody activity for themselves as well as for Moloney-HIX MCF cells, but the activity for the homologous 247 MCF MuLV-infected cells was only slightly reduced, leaving significant nonabsorbable activity. Conversely, absorption with MCF 247 cells completely eliminated the activity for all three MCF cells. Thus AKR-247 MCF possessed three MCF type-determinants as defined by the 827 serum, the Akv-2 congenic MCF possessed two, and Moloney-HIX MCF only one. Absorptions with Additional MuLVs to Further Define MCF Type Specificities. The absorption data presented above led to the conclusion that MCF MuLVs induce cellsurface antigens specific for ecotropic, xenotropic, and apparently MCF MuLVs. However, because the MCF viruses are most likely env gene recombinants between A K R eeotropic and as yet unidentified xenotropic MuLVs, it was possible that the MCF type specificities are distinct xenotropic M u L V specificities of the parental viruses. Absorption of anti-AKR-247 MCF serum was performed with a variety of xenotropic MuLV-infected cells to look at this possibility. Table IV summarizes these results. Absorption of the antiserum with a combination of ecotropic MuLV-infected cells and cells infected with xenotropic viruses of AKR, BALB/c, NZB, or NIH Swiss origin eliminated the reactions for themselves and for each other but did not significantly reduce the MCF activity. This showed that the xenotropic specificities were cross-reactive and that the MCF specificities were not associated with any of the xenotropic viruses tested. This confirmed that the MCFspecific activities do, indeed, detect new and distinct antigenic determinants. Antigenic Relationships between MCF Viruses Isolated from Different Sources. The serological relationship between different MCF isolates was further studied. Absorption of diluted (1:80) AKR-247 MCF antiserum with cells infected with various MCF viruses and then testing the absorbed serum on MCF-infected cells gave the results tabulated in Table V. Identical results were obtained with antiserum that had been preabsorbed with a combination of ecotropic (AKR2a) and xenotropic (BALB-IU-1) MuLVs, showing that only MCF type specificities were involved. Again, three MCF subspecificities were defined with this expanded panel. One specificity typed the MCF antigenic determinant(s) on Moloney-HIX MCF virusinfected cells, which was found to be shared with the Friend MCF virus. This was shown by the complete absorption of the antibody activity by Friend and MoloneyH I X MCF cells for each other but not for any other MCF cell. We operationally designated this determinant(s) as MCFA-1 (for MCF Antigen 1). The activity that was not absorbed by Friend or Moloney MCF viruses was further divisible into two
708
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS TABLE IV
Effect of Absorption with Combinations of AKR Ecotropic and Various Xenotropic MuL V-Infected Cells on Anti-AKR-247 MCF Serum Activities Cells used for absorption*
Residual antiserum activity for cells* infected with these MuLVs AKR2a ecotropic
Virus type
AKR6 xenotropic
Strain Antiserum 1:10
None None Ecotr AKR2a Ecotr + AKR2a + AKR6 Xtr AKR2a + AKR40 AKR2a + BALB-IU-
BALBIU- 1 xenotropic
NZB- AT124 Akv-2- AKRIU-3 xenoC26 247 x e n o - t r o p i c MCF MCF tropic
AntiAntiAntiAntiAntiAntis e r u m s e r u m s e r u m s e r u m s e r u m serum 1:10 1:10 1:10 1:10 1:100 1:100
90:~ 0 0
98 95 0
100 98 0
100 96 0
93 91 0
89 85 57
100 98 82
0 0
0 0
0 0
0 0
0 0
43 50
93 77
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
52 60 0 0
94 97 76 0
1
AKR2a + NZB-IU-3 AKR2a + AT124 MCF
Akv-2-C26 AKR-247
* Ecotropic MuLV was grown in NIH/3T3 cells. Xenotropic and MCF MuLVs were grown in mink lung cells (CCL 64). :~Percentage of cells fluorescing. a d d i t i o n a l M C F sub-specificities, designated M C F A - 2 a n d MCFA-3. O n e set of M C F M u L V s , i n c l u d i n g A K R - 1 3 , Akv-2-C34, Akv-2-C26, Akv-l-C44, a n d C58-C301-45, were M C F A - 1 +, M C F A - 2 +, M C F A - 3 - , in that they were able to completely absorb a n t i b o d y activity for each other a n d for the F r i e n d - M o l o n e y MCFs, b u t could not absorb all activity for a third group of M C F viruses (AKR-247, Akv-l-C36, Fg-C207647, C58-C298-48, a n d CB208). This latter group of M C F strains completely absorbed the activity for all M C F viruses tested, a n d are considered to express all three M C F A d e t e r m i n a n t s . As cell-absorption controls, F r i e n d ecotropic a n d wild mouse a m p h o tropic virus-infected cells did not absorb a n y of the a n t i s e r u m activities for M C F virus-infected cells. Other M C F Antisera. Because the MCF-specific antigens were d e f n e d with a single a n t i s e r u m (827), a second a n t i s e r u m raised against A K R - 2 4 7 (serum # R 2 5 4 6 1 ) a n d a serum prepared against a n o t h e r A K R M C F virus (AKR-13) were tested for their M u L V specificity. Similar to a n t i s e r u m 827, a n t i - A K R - 2 4 7 M C F serum R25461 d i s c r i m i n a t e d M C F A - 1 , -2, a n d -3 as well as type-specificities for ecotropic a n d xenotropic M u L V s (data not shown). T a b l e V I shows the absorption results o b t a i n e d with a n t i - A K R - 1 3 M C F serum R25415. Like A K R - 2 4 7 antiserum, A K R - 1 3 a n t i s e r u m c o n t a i n e d a n ecotropic M u L V specificity (absorbable by A K R ecotropic M u L V - i n f e c t e d cells b u t not by xenotropic M u L V ) , a xenotropic M u L V specificity (absorbable by xenotropic M u L V - i n f e c t e d cells b u t not by ecotropic-infected cells), a n d M C F type-specificities (absorbed by M C F cells b u t not by cells infected with ecotropic, xenotropic, or a c o m b i n a t i o n of eco- a n d xenotropic MuLVs). This antiserum, however, did not distinguish between
CLOYD,
HARTLEY,
AND
709
ROWE
TABLE V
Anti-AKR-247 M C F Serum Specificities for Various M C F MuL V-Infected Cell Lines Residual antiserum activity* for mink cells infected with these M C F M u L V s Cells antiserum absorbed with
Friend
Mol.HIX
AKR-13 Akv-2-C34
C58Akv-2-C26 Akv-l-C44 C301-45
C58Akv-l-C36 Fg-C207C298-48 647
CB208
A K R 247
88:~
93
I00
I00
92
100
98
92
100
100
100
100
Friend M C F MoI.-HIX M C F
0 0
0 0
87 52
81 62
31 54
98 51
91 28
80 82
99 60
100 90
83 69
100 79
AKR-13 M C F Akv-2-C34 M C F Akv-2-C26 M C F Akv-l-C44 M C F C58-C301-45 M C F
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 O 0 0
85 95 73 78 82
65 63 78 74 80
85 90 83 81 93
42 40 40 38 42
9O
C58-C298-48 M C F Akv-]-C36 M C F Fg-C207-647 CB208 M C F AKR-247 M C F
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
89 79
88 92
93 94
97 98
89 90
19O 100
90 98
90 92
95 99
I00
94 100
19O 19O
None
Friend Ecotr. 4070A
19O
95 86 82 78
* Antiserum dilution 1:80. Percent cells fluorescing. TABLE
VI
Activity of Anti-AKR-13 M C F Serum for MuL V-Infected Cells after Absorption with Various MuL V Cells Cells used for absorption Virus
Strain
Xtr. Ecotr. + MCF
Xtr.
None AKR2a NZB-IU-3 AKR2a + NZB-IU-3 Mol.-HIX AKR-13 AKR-247 Akv-l-C36 Akv-2-C34
NZB-
IU-3 xenotropic
Mol.HIX MCF
AKR- 13 MCF
AKR247 MCF
Akv-lC36 MCF
Akv-2C34 MCF
49:~ 0 45 0
40 22 0 0
100 77 40 36
100 94 98 89
100 92 96 80
100 98 98 92
100 99 98 97
48 0 0 0 0
0
0
0 0 0 0
79 0 0 0 0
87 0 0 0 0
90 0 0 0 0
91
0 0 0 0
ARK2a
type None Ecotr.
Residual antiserum activity* for cells infected with these MuLVs
eeotropic
0 0 0 0
* Antiserum dilution 1:60. :]: Percentage of cells fluorescing. t h e d i f f e r e n t n a t u r a l l y o c c u r r i n g M C F viruses (those n o t o f F r i e n d or M o l o n e y origin), d e m o n s t r a t i n g t h a t it d i d n o t c o n t a i n t h e t h i r d M C F specificity ( M C F A - 3 ) . T h i s was e x p e c t e d , b e c a u s e t h e d a t a in T a b l e V i n d i c a t e d t h a t A K R - 1 3 M C F l a c k e d t h a t determinant. These absorptions showed that the major ecotropic, xenotropic, and MCF type specificities a s s o c i a t e d w i t h A K R M C F M u L V s w e r e n o t p e c u l i a r to o n e a n t i s e r u m a n d t h a t d i f f e r e n t M C F isolates a p p e a r to i n d u c e s i m i l a r sets o f a n t i g e n s o n cells.
710
RECOMBINANTMURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
Discussion The purpose of this study was twofold: (a) to characterize reagent antisera made against A K R MCF MuLVs and (b) conversely, to characterize the cell-surface antigens induced by naturally occurring mouse MCF viruses. Sera against A K R MCF viruses were prepared in a heterologous species because such sera would be likely to recognize multiple viral antigenic markers. The use of rabbit cells for growing the immunizing virus allowed virus-specific antiserum to be made without the complication of also raising strong antiheterologous species reactivity. The absorption data clearly demonstrated that a number of distinct virus-associated antigens were indeed identifiable on cells infected with the cloned MCF viruses. These antigens included type-specific determinants of ecotropic and xenotropic MuLVs, as well as three new antigens apparently specific for MCF viruses. From the high titers of reactive antibodies that some of the new MCF type-specific antigens elicited, these particular antigens should probably be considered major determinants of MCF viruses. The MCF-specific antigens, which we operationally designate as MCFA-1, MCFA2 and MCFA-3, were unequally distributed among various MCF M u L V isolates. MCFA-1 was induced by all MCF viruses, including Friend and Moloney-HIX MCF viruses. MCFA-2 was associated with all other MCF viruses tested (those not of Friend or Moloney origin), whereas the third, MCFA-3, was induced by about one-half of the MCF M u L V isolates. The latter included strains from AKR (AKR-247); from NFS mice congenic for virus-inducing loci from AKR, C58, and C3H/Fg; and from BALB/c mice. It is noteworthy that other MCF isolates from similar mice did not elicit MCFA-3. At present it does not appear that the presence or absence of the MCFA-3 specificity can be correlated with any biological property of the viruses. In particular, there was no correlation between MCFA-3 and the leukemogenic capacity of these viruses (M. W. Cloyd, J. W. Hartley, W. P. Rowe, unpublished data). The production of antibodies against xenotropic- and ecotropic-specific cell-surface antigenic determinants by the animals immunized with cloned MCF viruses provides further evidence that these viruses are recombinants between ecotropic and xenotropic MuLVs. Because recombination appears to have occurred in the e n v gene region of MCF viruses (1-4), and because the techniques used here focused on cell-surface antigens, it seems likely the MCF type-specific antigens detected in this study are gp70 antigenic determinants. We were not able to discern this conclusively owing to the lack of purified AKR MCF viral components in sufficient quantities for absorption. However, it has been shown that antisera raised in the manner of those in this study predominantly contain anti-gp70 activity (9). In addition, because the MCF viruses appear to be gp70 recombinants between two recognized classes of MuLV, the presence of new antigenic determinants distinct from those of the putative parental viruses was not expected. One possibility could be that conformational or primary amino acid changes occurred in gp70 molecules after recombination. Another possibility is that these new antigenic determinants may be associated with an endogenous parental virus that is normally unexpressed and whose antigenic composition is unknown. Further studies are needed to clarify this. It should be noted that in another recent study MCF type-specific determinants were found in association with the gp70 molecule of Friend MCF MuLV (14). An attempt was made to correlate the MCF-associated antigens described herein
CLOYD, HARTLEY, AND ROWE
711
with other defined MuLV-associated cell surface antigens. The recently described G¢~a~sL2) MCF-associated antigen (15) was of particular importance. G(AKSI.~)has been associated with AKR-247, AKR-13, and Akv-1-C36 M C F viruses but was not induced by A k v - 2 - C 3 4 or C58 congenic MCF viruses. This pattern did not correlate with our M C F specificities (MCFA-3 was induced by AKR-247, A k v - l - C 3 6 , and C58-C298-48, but not by AKR-13; MCFA-1 and 2 were induced by all of these viruses). A K R MCF M u L V s have also been shown to induce Gix (associated with either ecotropic [16] or xenotropic [17] MuLVs), GmADAX~ (18) (associated with ecotropic MuLVs), and G~ER~) (associated with xenotropic MuLVs) (15). Work is in progress to determine if any of these antigens are those defined by our sera. With these defined reagents it should now be possible to more precisely elucidate the expression of different virus genomes in the mouse and to possibly gain more insight into the roles that they may play in leukemogenesis. In particular, attempts to detect the MCF-specific antigens in various preneoplastic and neoplastic tissues of mice will be of much interest. Summary Distinct type-specific antigens were detected on cells infected with cloned mink cell focus-inducing (MCF) murine leukemia viruses by means of cell surface immunofluorescence absorption assays with rabbit antisera raised against naturally-occurring A K R M C F viruses. The MCF type-specific antibodies were present in high titer and not absorbable by cells infected with ecotropic, xenotropic, or wild mouse amphotropic murine leukemia viruses, or combinations of ecotropic and xenotropic viruses. Three M C F subtype-specific reactions were identified. One subspecificity (operationally designated MCFA-1) defined antigenic determinant(s) distributed among M C F viruses in general. Another (MCFA-2) specified determinant(s) induced by all naturally occurring M C F isolates not of Friend or Moloney origin. A third subspecificity (MCFA-3) was induced by some MCF isolates, and not by others; the presence of this antigen did not correlate with the source of any presently known biological property of the viruses. In addition, type-specific antigenic determinants of ecotropic and xenotropic murine leukemia viruses were expressed on MCF virus-infected cells. The serological profile of MCF viruses thus supports the contention that they are env gene recombinants between ecotropic and xenotropic murine leukemia viruses. However, new, distinct MCF-specific determinants are also generated, and these could be useful markers in studying M C F viruses. Received for publication 27 November 1978.
References 1. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Natl. Acad. Sci. U. S. A. 74:789. 2. Elder, J. H., J. W. Gautsch, F. C. Jensen, R. A. Lerner, J. W. Hartley, and W. P. Rowe. 1977. Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recomhinants. Proc. Natl. Acad. Sci. U. S. A. 74:4676. 3. Rommelaere, J., D. V. Failer, and N. Hopkins. 1978. Characterization and mapping of RNase Tl-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses. Proc. Natl. Acad. Sci. U. S. A. 75:495.
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RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
4. Fischinger, P. J., S. Nomura, and D. P. Bolognesi. 1975. A novel murine oncornavirus with dual eco- and xenotropic properties. Proc. Natl. Acad. Sci. U. S. A. 72:5150. 5. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack host-range restriction for murine leukemia viruses. Virology. 65:128. 6. Todaro, G. J., P. Arnstein, W. P. Parks, E. H. Lennette, and R. J. Huebner. 1973. A typeC virus in human rhabdomyosarcoma cells after inoculation into NIH Swiss mice treated with anti-thymocyte serum. Proc. Natl. Acad. Sci. U. S. A. 70:859. 7. Hartley, J. W., and W. P. Rowe. 1976. Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.J. Virol. 19:19. 8. Rasheed, S., M. B. Gardner, and E. Chan. 1976. Amphotropic host range of naturally occurring wild mouse leukemia viruses. J. Virol. 19:13. 9. Morse, H. C. III, T. M. Chused, M. Boehn-Truitt, B. J. Mathieson, S. O. Sharrow, and J. W. Hartley. 1979. XenCSA: a cell surface antigen related to the major glycoprotein (gp70) of xenotropic murine leukemia virus. J. Immunol. In press. 10. Phillips, C. A., J. L. Melnick, and M. Burkhardt. 1966. Isolation, propagation, and neutralization of rubella virus in cultures of rabbit cornea (SIRC) cells. Proc. Soc. Exp. Biol. Med. 122:783. 11. Cloyd, M. W., and D. D. Bigner. 1977. Contained indirect viable-cell membrane immunofluorescence microassay for surface antigen analysis of cells infected with hazardous viruses. J. Clin. Microbiol. 5:86. 12. Cloyd, M. W., D. P. Bolognesi, and D. D. Bigner. 1977. Immunofluoreseent analysis of expression of the RNA tumor virus major glycoprotein, gp71, on surfaces of virus-producing murine and other mammalian species cell lines. Cancer Res. 37:922. 13. Barbacid, M., S. R. Tronick, and S. A. Aaronson. 1978. Isolation and characterization of an endogenous type C RNA virus of mink (MvlLu) cells. J. Virol. 25:129. 14. Ruscetti, S., D. Linemeyer, J. Feild, D. Troxler, and E. Scolnick. 1978. Type-specific radioimmunoassays for the gp70s of mink cell focus-inducing murine leukemia viruses: expression of a cross-reacting antigen in cells infected with the Friend strain of the spleen focus-forming virus. J. Exp. Med. 148:654. 15. Stockert, E., A. B. DeLeo, P. V. O'Donnell, Y. Obata, and L. J. Old. 1978. GIAKSL2~:a new cell surface antigen of the mouse related to the dual tropic MCF class of MuLV detected by naturally occurring antibody. J. Exp. Med. 149:200. 16. O'Donnell, P. V., and E. Stockert. 1976. Induction of Gm antigen and Gross cell surface antigen after infection by ecotropic and xenotropic murine leukemia viruses in vitro.J. Virol. 20:545. 17. Tung, J-S, P. V. O'Donnell, E. Fleissner, and E. A. Boyse. 1978. Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes. J. Exp. Med. 147:1280. 18. Obata, Y., E. Stockert, P. V. O'Donnell, S. Okubo, H. W. Snyder, Jr., and L.J. Old. 1978. GIRADA1): a new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus.J. Exp. Med. 147:1089.
ANTIGENS
RECOMBINANT
MINK
MURINE
ASSOCIATED
WITH
CELL FOCUS-INDUCING
LEUKEMIA
VIRUSES
BY MILES W. CLOYD,* JANET W. HARTLEY, AND WALLACE P. ROWE From the Laboratory of Viral Diseases, National Institute of Aller~ and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014 The endogenous retroviruses of mice, which exist as normal, inherited components of the mouse genome, are generally classified on the basis of their species host range as ecotropic (capable of infecting only mouse or rat cells), xenotropic (capable of infecting only heterologous species cells), or amphotropic (wild mouse viruses capable of infecting both mouse and heterologous species cells). Recently, a new class of murine leukemia virus (MuLV) 1 was described which has properties of both xenotropic and ecotropic viruses and which induces morphological changes in the mink lung cell line A T C C No. CCL-64 (1). Abundant evidence indicates that these viruses, designated mink cell focus-inducing (MCF) viruses, are env gene recombinants between ecotropic and xenotropic MuLVs (1-4). The emergence of MCF viruses from lymphoid tissue during the preleukemic or leukemic state may indicate that they are involved in some way in the pathogenesis of murine leukemia (1). To obtain more understanding about the natural history of M C F viruses, we have attempted to develop MCF-specific serological probes which could be applied in identification, classification, and characterization of in vivo expression of MCF viruses. In this report we describe the specificity of these reagents and the corresponding cellsurface antigens induced in cell cultures infected with M C F viruses. Reagent rabbit antisera prepared against A K R MCF-infected rabbit cells were able to distinguish major antigenic determinants specific for ecotropic, xenotropic, and three subclasses of MCF viruses. Materials and Methods Viruses and Cells. Naturally occurring MCF viruses were isolated from mice by plating mitomycin C-treated lymphoid cells as infectious centers onto CCL-64 mink cells as described previously (1). The viruses were adapted to cell-free passage in SC-1 (5) and/or mink lung cell cultures, and were carried through two cycles of limiting dilution purification. Table I lists the viruses and cells used in this study. Tests with Moloney MCF were done chiefly with a strain isolated by Dr. Akinori Ishimoto of this laboratory; however, the prototype recombinant virus from Moloney stocks, HIX (4), kindly supplied by Dr. Peter Fischinger, National Institutes of Health, was also used and was found to be serologically identical to this Moloney MCF isolate. HIX and Moloney MCF are therefore used interchangeably throughout this report. Most of * Recipient of a Fellowship from the Damon Runyon-Walter Winchell Cancer Fund, N. Y. 1 Abbreviations used in this paper: MuLV, murine leukemia virus; MCF, mink cell focus-inducing; env,
envelope; IUdR, 5-iododeoxyuridine; MiLV, mink leukemia virus; ecotr., ecotropic; Xtr., xenotropic; Amtr., amphotropic.
702
THE J O U R N A L OF E X P E R I M E N T A L MEDICINE • V O L U M E 149, 1979
703
CLOYD, HARTLEY, AND ROWE TABLE I Description of Murine Leukemia Viruses Used Class Ecotropic
Strain
Thymus of 2-mo-old AKR Mouse
AKRL1
Spleen, thymus, and nodes from spontaneous AKR leukemia. Tail extract of young Akv-I congenie mouse. NB-tropic virus isolated from mouse-passaged Moloney stock. NB-tropic virus isolated from BALB/c passed stock. BALB/C embryo cells treated with IUdR. Thymus of 2-mo-old AKR mouse. Thymus of 6-mo-old A K R mouse NZB-Q cell line treated with IUdR. Isolated by Todaro et al. (6) from an NIH Swiss mouse. IUdR-treated embryo cells of wild mouse from Lake Casitas (Calif.) area. Embryo cells of wild mouse from Lake Casitas (Calif.) (7, 8). Embryo cells of wild mouse from LaPuente (Calif.) area. Thymus of normal 6-mo-old A K R / J mouse (1). Thymus of 3-too-old A K R / J mouse that had received thymus graft from 6-mo-old A K R / J 1 mo previously. Spontaneous lymphoma of NFS mouse partially congenic for Akv-2. Spontaneous reticulum cell sarcoma of NFS mouse partially congenic for Akv-2. Spontaneous lymphoma of NFS mouse partially congenic for Akv-1. Spontaneous reticulum cell sarcoma of NFS mouse partially congenlc for Akv-l. Spontaneous reticulum cell sarcoma of NFS mouse partially congenic for a C58/Lw virus-inducing locus. Spontaneous Hodgkin's-type reticulum cell sarcoma of NFS mouse partially congenic for a C58/Lw virus-induclng locus. Spontaneous lymphoma of NFS mouse partially congenie for C3H/Fg virus-inducing locus Fgv-l. Pristane-induced plasmacytoma ofa BALB/c mouse. Moloney stock passed in BALB/c mice (A. Ishimoto, unpublished observations). IC-Strain of Moloney MuLV (4). Friend stock passed in BALB/c mice (A. Ishimoto, unpublished observations).
RS-18 MIT Moloney
Xenotropic
Friend BALB-IU-1 AKR6 AKR40 NZB-IU-3 AT124 Cas.E 1
Amphotropic
4070A 1504A
MCF
Source
AKR2a
AKR-247 AKR-13
Akv-2-C34* Akv-2-C26 Akv- l-C36 Akv- 1-C44 C58-C301-45
C58-C298-48
Fg-C207-647 CB208 Mol. MCF HIX Friend MCF
Grown In* NIH/3T3 FRE, SC-1 NIH/3T3 SC- 1 NIH/3T3 FRE, SC-1 Mink Mink Mink Mink Mink Mink Mink Mink SC-I Mink Mink
Mink Mink Mink Mink Mink
Mink
Mink Mink Mink Mink Mink
* Cells include NIH/3T3; Fisher rat embryo (FRE C12, from Dr. Edward Scolnick, NIH); SC-1 (a cloned line of wild mouse embryo cells); and mink lung cells (CCL64). :]: The C in the virus designation refers to its origin from a congenic mouse.
704
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
the MCF isolates were N-tropic. Exceptions include an isolate (CB208) from a BALB/c plasmacytoma, which was B-tropic; and Moloney, HIX, and Friend MCF viruses, which were NB-tropie. Mink cells replicating an endogenous mink C-type virus (MiLV) were kindly supplied by Dr. Raymond Gilden, Frederick Cancer Research Center, Ft. Detrick, Frederick, Md. All cells were grown in the Dulbecco-Vogt modification of Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal calf serum, 2mM glutamine, 250 U / m l penicillin, and 250 ~g/ml streptomycin. Antisera and SerologicalAssay. MCF antisera were raised in New Zealand white rabbits by a single intravenous injection of 2 × 10v MCF-infected SIRC cells as described elsewhere (9); SIRC is a tissue culture line of rabbit corneal cells (10). Anti-AKR-247 MCF serum 827 was obtained from a rabbit 3 wk after injection, precipitated with sodium sulfate, dialysed, and reconstituted at a four-fold concentration. It was kindly supplied by Dr. Thomas Chused, National Institutes of Health. Anti-AKR-247 MCF serum R25461 and anti-AKR-13 MCF serum R25415 were from pooled weekly bleedings taken 2-5 wk after injection. Control sera included pre-immune sera and an anti-SIRC cell serum, prepared in the manner described above using uninfected SIRC cells. An indirect live-cell immunofluorescenee assay described previously (11) was used, with slight modification, to score for antibody activity. Briefly, 1 × 105 freshly suspended cells were mixed with 50 #1 (total vol, 60/~1) of diluted rabbit antiserum in wells of a microtiter plate for 20 min at room temperature, rinsed, and then incubated similarly with 50/~1 of appropriately diluted fluorescein-eonjugated goat anti-rabbit IgG (Meloy Laboratories Inc., Springfield, Va.). After rinsing, the cells were examined under epi-UV illumination at 300 times magnification, and the percentage of cells showing surface fluorescence determined. Absorptions. To determine antibody specificity, qualitative absorptions were performed. The experimental design was to absorb with one set of antigens (virus-infected cells) and then test the absorbed antiserum on a panel of target cells. The procedure, described in detail elsewhere (12), entailed mixing, in duplicate, 5 X 10v freshly trypsinized tissue culture cells with 0.5 ml of antiserum diluted about fourfold less than the 50% staining point for the chosen target cell. After overnight incubation at 4°C, the sera were centrifuged; the serum from one tube was frozen, and the serum from the other was mixed with another 5 × 10 7 cells for a second absorption. The one- and two-times absorbed antisera were then tested on each target cell; if the second absorption did not significantly reduce the antibody activity (as determined by percent cells stained) below that seen after one absorption, the antiserum was considered completely absorbed. If the activity was significantly decreased with the second absorption but some activity remained, then an additional absorption was performed. Residual staining activity for a target cell was therefore interpreted as demonstrating that an antigenic determinant detected on that target cell was not present on the absorbing cell. All the absorption data reported herein were obtained with doubly absorbed antisera; three absorptions were not required. Results
Activity of Anti-AKR-247 MCF Serum for Cells Infected with Various MuL Vs.
Three antisera p r e p a r e d against A K R - 2 4 7 M C F - i n f e c t e d S I R C cells (antiserum 827, R25461, a n d a h y p e r i m m u n e serum, 48, which was not studied further) were t i t r a t e d in the m i c r o - i m m u n o f l u o r e s c e n c e test against various virus-infected a n d control cells to ascertain the extent o f their a n t i b o d y activity. All were c o m p a r a b l y active against the various M u L V - i n f e c t e d cells; the activity of a representative serum (827) is s u m m a r i z e d in T a b l e II. U n i n f e c t e d mink, SC-1, or S I R C cells, a n d m i n k cells r e p l i c a t i n g an e n d o g e n o u s m i n k C - t y p e virus (13) were not significantly reactive with this a n t i s e r u m , i.e., the 50% s t a i n i n g titers were between 1:2 a n d 1:5. Cells infected with M o l o n e y or F r i e n d ecotropic viruses were slightly reactive (50% titers of 21:10), a n d cells infected with A K R - t y p e ecotropic viruses gave 50% serum titers o f 1:60 to 1:75. T h e a n t i s e r u m was also significantly active against cells infected with xenotropic a n d a m p h o t r o p i c
705
CLOYD, HARTLEY, AND ROWE TABLE II Anti-AKR-247 MCF Serum (827) Activity for Cells Infected with Various C-Type Viruses Control cells
Mink SC-I SIRC Mink infected with MiLV
50%* titer 2 5 5
EcotroF" (cell)
50%* titer
MIT Moloney (FRE) MIT Moloney (SC- 1) Friend (Mink)
10 12 12
AKR2a (NIH/3T3) AKR2a (FRE)
60 65
AKR2a (SO-l)
75
50%* titer
Amphotropic virus (cell)
50%* Titer
MCF virus (cell)
50%*
ATI24 (Mink) AKR40 (Mink) AKR6 (Mink)
20 30 3O
4070A(Mink) 1504A(SO-1)
20 18
I00 250 250
NZB-IU-3 (Mink) BALB-IU-1 (Mink)
50 7O
Friend (Mink) Mol.-HIX (Mink) C58-C301-45 (Mink) Akv-2.C26 (Mink) C58-C298-48 (Mink) Akv-2-C34 (Mink) A K R I 3 (Mink) Akv-I-C44 (Mink) Akv- I-C36 (Mink) Fg-C207-647 (Mink) CB208 (Mink) AKR-247 (Mink)
Xenotropic virus (ceil)
titer
250 250 300 3OO 340 340 340 4OO 64O
* Reciprocal of antiserum dilution that stains 50% of the cells.
MuLVs; the highest titer for xenotropic-infected cells was that for the BALB-IU-1 strain (50% titer 1:70). Lesser activities were found for NZB, AKR, and NIH Swiss xenotropic viruses and for wild mouse amphotropic MuLV-infected cells. The antiserum displayed the greatest activity for MCF MuLV-infected cells (50% titers ranging from 1:100 to 1:640), with cells infected with the homologous AKR-247 MCF virus giving the highest titer. Pre-immune and rabbit anti-SIRC cell control antisera were also tested on approximately one-half of the cells and were found not to be significantly reactive, giving 50% staining titers between 1:1 and 1:3 (data not shown). In neutralization tests, the MCF-247 antiserum displayed similar activities, having slight neutralization titers for ecotropic and xenotropic MuLVs (< 1:20) but significant activity for MCF viruses (1:40-1 : 160). From these studies, it was concluded that the anti-AKR-247 MCF serum was reacting specifically with MuLV-associated cell-surface antigens and that the antiserum most likely contained multiple MuLV specificities. Qualitative Absorptions to Determine Serological Specificity. The specificity of the antibody activity for the various MuLV-producing cell lines was analysed by absorptions. Antiserum 827 was absorbed with various MuLV-infected cells and then tested for activity on various virus-infected cells (Table III). Absorbing with uninfected mink, SIRC, or N I H / 3 T 3 cells did not appreciably reduce the antiserum activity (at a 1:10 dilution) for ecotropic, amphotropic, or xenotropic MuLV-infected cells or (at 1:801:100 dilutions) for MCF cells. These absorptions not only served as absorption controls, showing that nonspecific absorption was not involved, but also demonstrated that cellular, nonviral antigens were not being detected. Absorption with cells infected with AKR ecotropic MuLV removed only the antibody activity for the homologous target cells (AKR2a); the activities for cells infected with amphotropic, xenotropic, and three MCF M u L V strains were but slightly reduced. In addition to this AKR ecotropic MuLV isolate, two other ecotropic viruses from AKR (AKRL1 and RS-18) were tested for their absorption efficiency; the three strains absorbed identically and were completely cross reactive (data not shown). Absorptions with Moloney ecotropic virus-infected cells slightly diminished
706
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS TABLE III
Specificity of Anti-AKR-24 7 MCF Serum for MuL V-Infected Cells as Defined by Absorptions Cells used for absorption*
Virus type
None
Ecotr. Amtr. Xtr. Ecotro + Xtr MCF
Residual antiserum activity for cells* infected with these MuLVs AKR2a ecotropic
4070A amphotropic
Strain
BALBIU-1 xenotropic
Antise- AntiseAntiserum rum rum l:10 1:10 1:10 None Mink lung SIRC NIH/3T3 AKR2a Mol. 4070A BALB-IU-1 AKR2a + BALB-IU-1 Mol. + BALB-IU-1 Mol.-HIX
Akv-2-C26 AKR-247
Mol.HIX MCF
Akv-2C26 MCF
AKR247 MCF
Antise- Antise- Antiserum rum rum 1:80 l:100 1:100
91:]: 90 90 90 0 83 90 90 0
69 62 60 58 57 51 0 0 0
100 100 100 100 82 80 50 0 0
90 89 86 83 81 58 90 62 51
92 90 89 91 85 88 90 83 57
100 100 100 100 98 100 100 95 72
75 80 0 0
0 0 0 0
0 0 0 0
30 0 0 0
NT§ 54 0 0
84 79 76 0
* MCF, amphotropic, and xenotropic MuLVs were grown in mink lung cells (CCL 64). Ecotropic MuLVs were grown in NIH/3T3 cells. :~Percentage of cells fluorescing. § NT, not tested. the a n t i s e r u m activity for M o l o n e y - H I X M C F cells a n d did not significantly reduce a n y other activities, i n c l u d i n g that for A K R ecotropic M u L V . A b s o r b i n g with cells infected with wild mouse a m p h o t r o p i c virus e l i m i n a t e d the a n t i b o d y activity for the homologous target cells (4070A) a n d reduced by one-half the activity for BALB-IU-1 xenotropic M u L V cells, b u t it did not affect the activity for cells infected with A K R ecotropic or M C F viruses. Absorption with cells infected with B A L B / c xenotropic M u L V (BALB-IU-1) e l i m i n a t e d the a n t i b o d y activity for both a m p h o t r o p i c a n d xenotropic M u L V - i n f e c t e d cells, b u t only slightly reduced the activity for ecotropic- or M C F - i n f e c t e d cells. It thus appears that the a m p h o t r o p i c M u L V specificity detected with the 827 serum is reactive with a n a n t i g e n shared with xenotropic viruses. T h e a n t i s e r u m therefore c o n t a i n e d a specificity for ecotropic M u L V , not removed by absorption with xenotropic or a m p h o t r o p i c M u L V s , a n d a distinct specificity for x e n o t r o p i c - a m p h o t r o p i c M u L V s , not removed by absorption with ecotropic M u L V infected cells. T h e activity for M C F cells, not b e i n g absorbed by either amphotropic, xenotropic, or A K R ecotropic M u L V - i n f e c t e d cells, was clearly not one of these respective specificities. Also, as shown in T a b l e III a n d e x a m i n e d in more detail in a later section, this activity was not removed by absorptions with mixtures of ecotropic a n d xenotropic M u L V - i n f e c t e d cells. This indicated that a significant M C F - t y p e specificity was present in this antiserum.
CLOYD, HARTLEY, AND ROWE
707
Absorptions were also performed with the MCF MuLV-infected cells to determine if they expressed the ecotropic and xenotropic MuLV-specific determinants and to explore the relationships between the different MCF isolates. Ceils replicating MCF MuLVs of A K R and Akv-2 congenic origin were able to absorb completely both the A K R ecotropic and xenotropic type-specificities (Table III), showing that they do express both sets of determinants. Cells infected with Moloney-HIX MCF M u L V demonstrated a xenotropic component, but they were not cross-reactive with A K R ecotropic virus; this further demonstrates that there are significant differences between Moloney and A K R ecotropic components. Differential absorption of MCF activities by different MCF cells showed that at least three distinct MCF type-specific determinants could be discriminated. Moloney-HIX MCF was able to completely absorb the antibody activity for itself, but partial activity remained for two other MCF isolates. Cells replicating Akv-2-C26 MCF were able to completely absorb the antibody activity for themselves as well as for Moloney-HIX MCF cells, but the activity for the homologous 247 MCF MuLV-infected cells was only slightly reduced, leaving significant nonabsorbable activity. Conversely, absorption with MCF 247 cells completely eliminated the activity for all three MCF cells. Thus AKR-247 MCF possessed three MCF type-determinants as defined by the 827 serum, the Akv-2 congenic MCF possessed two, and Moloney-HIX MCF only one. Absorptions with Additional MuLVs to Further Define MCF Type Specificities. The absorption data presented above led to the conclusion that MCF MuLVs induce cellsurface antigens specific for ecotropic, xenotropic, and apparently MCF MuLVs. However, because the MCF viruses are most likely env gene recombinants between A K R eeotropic and as yet unidentified xenotropic MuLVs, it was possible that the MCF type specificities are distinct xenotropic M u L V specificities of the parental viruses. Absorption of anti-AKR-247 MCF serum was performed with a variety of xenotropic MuLV-infected cells to look at this possibility. Table IV summarizes these results. Absorption of the antiserum with a combination of ecotropic MuLV-infected cells and cells infected with xenotropic viruses of AKR, BALB/c, NZB, or NIH Swiss origin eliminated the reactions for themselves and for each other but did not significantly reduce the MCF activity. This showed that the xenotropic specificities were cross-reactive and that the MCF specificities were not associated with any of the xenotropic viruses tested. This confirmed that the MCFspecific activities do, indeed, detect new and distinct antigenic determinants. Antigenic Relationships between MCF Viruses Isolated from Different Sources. The serological relationship between different MCF isolates was further studied. Absorption of diluted (1:80) AKR-247 MCF antiserum with cells infected with various MCF viruses and then testing the absorbed serum on MCF-infected cells gave the results tabulated in Table V. Identical results were obtained with antiserum that had been preabsorbed with a combination of ecotropic (AKR2a) and xenotropic (BALB-IU-1) MuLVs, showing that only MCF type specificities were involved. Again, three MCF subspecificities were defined with this expanded panel. One specificity typed the MCF antigenic determinant(s) on Moloney-HIX MCF virusinfected cells, which was found to be shared with the Friend MCF virus. This was shown by the complete absorption of the antibody activity by Friend and MoloneyH I X MCF cells for each other but not for any other MCF cell. We operationally designated this determinant(s) as MCFA-1 (for MCF Antigen 1). The activity that was not absorbed by Friend or Moloney MCF viruses was further divisible into two
708
RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS TABLE IV
Effect of Absorption with Combinations of AKR Ecotropic and Various Xenotropic MuL V-Infected Cells on Anti-AKR-247 MCF Serum Activities Cells used for absorption*
Residual antiserum activity for cells* infected with these MuLVs AKR2a ecotropic
Virus type
AKR6 xenotropic
Strain Antiserum 1:10
None None Ecotr AKR2a Ecotr + AKR2a + AKR6 Xtr AKR2a + AKR40 AKR2a + BALB-IU-
BALBIU- 1 xenotropic
NZB- AT124 Akv-2- AKRIU-3 xenoC26 247 x e n o - t r o p i c MCF MCF tropic
AntiAntiAntiAntiAntiAntis e r u m s e r u m s e r u m s e r u m s e r u m serum 1:10 1:10 1:10 1:10 1:100 1:100
90:~ 0 0
98 95 0
100 98 0
100 96 0
93 91 0
89 85 57
100 98 82
0 0
0 0
0 0
0 0
0 0
43 50
93 77
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
52 60 0 0
94 97 76 0
1
AKR2a + NZB-IU-3 AKR2a + AT124 MCF
Akv-2-C26 AKR-247
* Ecotropic MuLV was grown in NIH/3T3 cells. Xenotropic and MCF MuLVs were grown in mink lung cells (CCL 64). :~Percentage of cells fluorescing. a d d i t i o n a l M C F sub-specificities, designated M C F A - 2 a n d MCFA-3. O n e set of M C F M u L V s , i n c l u d i n g A K R - 1 3 , Akv-2-C34, Akv-2-C26, Akv-l-C44, a n d C58-C301-45, were M C F A - 1 +, M C F A - 2 +, M C F A - 3 - , in that they were able to completely absorb a n t i b o d y activity for each other a n d for the F r i e n d - M o l o n e y MCFs, b u t could not absorb all activity for a third group of M C F viruses (AKR-247, Akv-l-C36, Fg-C207647, C58-C298-48, a n d CB208). This latter group of M C F strains completely absorbed the activity for all M C F viruses tested, a n d are considered to express all three M C F A d e t e r m i n a n t s . As cell-absorption controls, F r i e n d ecotropic a n d wild mouse a m p h o tropic virus-infected cells did not absorb a n y of the a n t i s e r u m activities for M C F virus-infected cells. Other M C F Antisera. Because the MCF-specific antigens were d e f n e d with a single a n t i s e r u m (827), a second a n t i s e r u m raised against A K R - 2 4 7 (serum # R 2 5 4 6 1 ) a n d a serum prepared against a n o t h e r A K R M C F virus (AKR-13) were tested for their M u L V specificity. Similar to a n t i s e r u m 827, a n t i - A K R - 2 4 7 M C F serum R25461 d i s c r i m i n a t e d M C F A - 1 , -2, a n d -3 as well as type-specificities for ecotropic a n d xenotropic M u L V s (data not shown). T a b l e V I shows the absorption results o b t a i n e d with a n t i - A K R - 1 3 M C F serum R25415. Like A K R - 2 4 7 antiserum, A K R - 1 3 a n t i s e r u m c o n t a i n e d a n ecotropic M u L V specificity (absorbable by A K R ecotropic M u L V - i n f e c t e d cells b u t not by xenotropic M u L V ) , a xenotropic M u L V specificity (absorbable by xenotropic M u L V - i n f e c t e d cells b u t not by ecotropic-infected cells), a n d M C F type-specificities (absorbed by M C F cells b u t not by cells infected with ecotropic, xenotropic, or a c o m b i n a t i o n of eco- a n d xenotropic MuLVs). This antiserum, however, did not distinguish between
CLOYD,
HARTLEY,
AND
709
ROWE
TABLE V
Anti-AKR-247 M C F Serum Specificities for Various M C F MuL V-Infected Cell Lines Residual antiserum activity* for mink cells infected with these M C F M u L V s Cells antiserum absorbed with
Friend
Mol.HIX
AKR-13 Akv-2-C34
C58Akv-2-C26 Akv-l-C44 C301-45
C58Akv-l-C36 Fg-C207C298-48 647
CB208
A K R 247
88:~
93
I00
I00
92
100
98
92
100
100
100
100
Friend M C F MoI.-HIX M C F
0 0
0 0
87 52
81 62
31 54
98 51
91 28
80 82
99 60
100 90
83 69
100 79
AKR-13 M C F Akv-2-C34 M C F Akv-2-C26 M C F Akv-l-C44 M C F C58-C301-45 M C F
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 O 0 0
85 95 73 78 82
65 63 78 74 80
85 90 83 81 93
42 40 40 38 42
9O
C58-C298-48 M C F Akv-]-C36 M C F Fg-C207-647 CB208 M C F AKR-247 M C F
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
89 79
88 92
93 94
97 98
89 90
19O 100
90 98
90 92
95 99
I00
94 100
19O 19O
None
Friend Ecotr. 4070A
19O
95 86 82 78
* Antiserum dilution 1:80. Percent cells fluorescing. TABLE
VI
Activity of Anti-AKR-13 M C F Serum for MuL V-Infected Cells after Absorption with Various MuL V Cells Cells used for absorption Virus
Strain
Xtr. Ecotr. + MCF
Xtr.
None AKR2a NZB-IU-3 AKR2a + NZB-IU-3 Mol.-HIX AKR-13 AKR-247 Akv-l-C36 Akv-2-C34
NZB-
IU-3 xenotropic
Mol.HIX MCF
AKR- 13 MCF
AKR247 MCF
Akv-lC36 MCF
Akv-2C34 MCF
49:~ 0 45 0
40 22 0 0
100 77 40 36
100 94 98 89
100 92 96 80
100 98 98 92
100 99 98 97
48 0 0 0 0
0
0
0 0 0 0
79 0 0 0 0
87 0 0 0 0
90 0 0 0 0
91
0 0 0 0
ARK2a
type None Ecotr.
Residual antiserum activity* for cells infected with these MuLVs
eeotropic
0 0 0 0
* Antiserum dilution 1:60. :]: Percentage of cells fluorescing. t h e d i f f e r e n t n a t u r a l l y o c c u r r i n g M C F viruses (those n o t o f F r i e n d or M o l o n e y origin), d e m o n s t r a t i n g t h a t it d i d n o t c o n t a i n t h e t h i r d M C F specificity ( M C F A - 3 ) . T h i s was e x p e c t e d , b e c a u s e t h e d a t a in T a b l e V i n d i c a t e d t h a t A K R - 1 3 M C F l a c k e d t h a t determinant. These absorptions showed that the major ecotropic, xenotropic, and MCF type specificities a s s o c i a t e d w i t h A K R M C F M u L V s w e r e n o t p e c u l i a r to o n e a n t i s e r u m a n d t h a t d i f f e r e n t M C F isolates a p p e a r to i n d u c e s i m i l a r sets o f a n t i g e n s o n cells.
710
RECOMBINANTMURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
Discussion The purpose of this study was twofold: (a) to characterize reagent antisera made against A K R MCF MuLVs and (b) conversely, to characterize the cell-surface antigens induced by naturally occurring mouse MCF viruses. Sera against A K R MCF viruses were prepared in a heterologous species because such sera would be likely to recognize multiple viral antigenic markers. The use of rabbit cells for growing the immunizing virus allowed virus-specific antiserum to be made without the complication of also raising strong antiheterologous species reactivity. The absorption data clearly demonstrated that a number of distinct virus-associated antigens were indeed identifiable on cells infected with the cloned MCF viruses. These antigens included type-specific determinants of ecotropic and xenotropic MuLVs, as well as three new antigens apparently specific for MCF viruses. From the high titers of reactive antibodies that some of the new MCF type-specific antigens elicited, these particular antigens should probably be considered major determinants of MCF viruses. The MCF-specific antigens, which we operationally designate as MCFA-1, MCFA2 and MCFA-3, were unequally distributed among various MCF M u L V isolates. MCFA-1 was induced by all MCF viruses, including Friend and Moloney-HIX MCF viruses. MCFA-2 was associated with all other MCF viruses tested (those not of Friend or Moloney origin), whereas the third, MCFA-3, was induced by about one-half of the MCF M u L V isolates. The latter included strains from AKR (AKR-247); from NFS mice congenic for virus-inducing loci from AKR, C58, and C3H/Fg; and from BALB/c mice. It is noteworthy that other MCF isolates from similar mice did not elicit MCFA-3. At present it does not appear that the presence or absence of the MCFA-3 specificity can be correlated with any biological property of the viruses. In particular, there was no correlation between MCFA-3 and the leukemogenic capacity of these viruses (M. W. Cloyd, J. W. Hartley, W. P. Rowe, unpublished data). The production of antibodies against xenotropic- and ecotropic-specific cell-surface antigenic determinants by the animals immunized with cloned MCF viruses provides further evidence that these viruses are recombinants between ecotropic and xenotropic MuLVs. Because recombination appears to have occurred in the e n v gene region of MCF viruses (1-4), and because the techniques used here focused on cell-surface antigens, it seems likely the MCF type-specific antigens detected in this study are gp70 antigenic determinants. We were not able to discern this conclusively owing to the lack of purified AKR MCF viral components in sufficient quantities for absorption. However, it has been shown that antisera raised in the manner of those in this study predominantly contain anti-gp70 activity (9). In addition, because the MCF viruses appear to be gp70 recombinants between two recognized classes of MuLV, the presence of new antigenic determinants distinct from those of the putative parental viruses was not expected. One possibility could be that conformational or primary amino acid changes occurred in gp70 molecules after recombination. Another possibility is that these new antigenic determinants may be associated with an endogenous parental virus that is normally unexpressed and whose antigenic composition is unknown. Further studies are needed to clarify this. It should be noted that in another recent study MCF type-specific determinants were found in association with the gp70 molecule of Friend MCF MuLV (14). An attempt was made to correlate the MCF-associated antigens described herein
CLOYD, HARTLEY, AND ROWE
711
with other defined MuLV-associated cell surface antigens. The recently described G¢~a~sL2) MCF-associated antigen (15) was of particular importance. G(AKSI.~)has been associated with AKR-247, AKR-13, and Akv-1-C36 M C F viruses but was not induced by A k v - 2 - C 3 4 or C58 congenic MCF viruses. This pattern did not correlate with our M C F specificities (MCFA-3 was induced by AKR-247, A k v - l - C 3 6 , and C58-C298-48, but not by AKR-13; MCFA-1 and 2 were induced by all of these viruses). A K R MCF M u L V s have also been shown to induce Gix (associated with either ecotropic [16] or xenotropic [17] MuLVs), GmADAX~ (18) (associated with ecotropic MuLVs), and G~ER~) (associated with xenotropic MuLVs) (15). Work is in progress to determine if any of these antigens are those defined by our sera. With these defined reagents it should now be possible to more precisely elucidate the expression of different virus genomes in the mouse and to possibly gain more insight into the roles that they may play in leukemogenesis. In particular, attempts to detect the MCF-specific antigens in various preneoplastic and neoplastic tissues of mice will be of much interest. Summary Distinct type-specific antigens were detected on cells infected with cloned mink cell focus-inducing (MCF) murine leukemia viruses by means of cell surface immunofluorescence absorption assays with rabbit antisera raised against naturally-occurring A K R M C F viruses. The MCF type-specific antibodies were present in high titer and not absorbable by cells infected with ecotropic, xenotropic, or wild mouse amphotropic murine leukemia viruses, or combinations of ecotropic and xenotropic viruses. Three M C F subtype-specific reactions were identified. One subspecificity (operationally designated MCFA-1) defined antigenic determinant(s) distributed among M C F viruses in general. Another (MCFA-2) specified determinant(s) induced by all naturally occurring M C F isolates not of Friend or Moloney origin. A third subspecificity (MCFA-3) was induced by some MCF isolates, and not by others; the presence of this antigen did not correlate with the source of any presently known biological property of the viruses. In addition, type-specific antigenic determinants of ecotropic and xenotropic murine leukemia viruses were expressed on MCF virus-infected cells. The serological profile of MCF viruses thus supports the contention that they are env gene recombinants between ecotropic and xenotropic murine leukemia viruses. However, new, distinct MCF-specific determinants are also generated, and these could be useful markers in studying M C F viruses. Received for publication 27 November 1978.
References 1. Hartley, J. W., N. K. Wolford, L. J. Old, and W. P. Rowe. 1977. A new class of murine leukemia virus associated with development of spontaneous lymphomas. Proc. Natl. Acad. Sci. U. S. A. 74:789. 2. Elder, J. H., J. W. Gautsch, F. C. Jensen, R. A. Lerner, J. W. Hartley, and W. P. Rowe. 1977. Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recomhinants. Proc. Natl. Acad. Sci. U. S. A. 74:4676. 3. Rommelaere, J., D. V. Failer, and N. Hopkins. 1978. Characterization and mapping of RNase Tl-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses. Proc. Natl. Acad. Sci. U. S. A. 75:495.
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RECOMBINANT MURINE LEUKEMIA VIRUS CELL-SURFACE ANTIGENS
4. Fischinger, P. J., S. Nomura, and D. P. Bolognesi. 1975. A novel murine oncornavirus with dual eco- and xenotropic properties. Proc. Natl. Acad. Sci. U. S. A. 72:5150. 5. Hartley, J. W., and W. P. Rowe. 1975. Clonal cell lines from a feral mouse embryo which lack host-range restriction for murine leukemia viruses. Virology. 65:128. 6. Todaro, G. J., P. Arnstein, W. P. Parks, E. H. Lennette, and R. J. Huebner. 1973. A typeC virus in human rhabdomyosarcoma cells after inoculation into NIH Swiss mice treated with anti-thymocyte serum. Proc. Natl. Acad. Sci. U. S. A. 70:859. 7. Hartley, J. W., and W. P. Rowe. 1976. Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.J. Virol. 19:19. 8. Rasheed, S., M. B. Gardner, and E. Chan. 1976. Amphotropic host range of naturally occurring wild mouse leukemia viruses. J. Virol. 19:13. 9. Morse, H. C. III, T. M. Chused, M. Boehn-Truitt, B. J. Mathieson, S. O. Sharrow, and J. W. Hartley. 1979. XenCSA: a cell surface antigen related to the major glycoprotein (gp70) of xenotropic murine leukemia virus. J. Immunol. In press. 10. Phillips, C. A., J. L. Melnick, and M. Burkhardt. 1966. Isolation, propagation, and neutralization of rubella virus in cultures of rabbit cornea (SIRC) cells. Proc. Soc. Exp. Biol. Med. 122:783. 11. Cloyd, M. W., and D. D. Bigner. 1977. Contained indirect viable-cell membrane immunofluorescence microassay for surface antigen analysis of cells infected with hazardous viruses. J. Clin. Microbiol. 5:86. 12. Cloyd, M. W., D. P. Bolognesi, and D. D. Bigner. 1977. Immunofluoreseent analysis of expression of the RNA tumor virus major glycoprotein, gp71, on surfaces of virus-producing murine and other mammalian species cell lines. Cancer Res. 37:922. 13. Barbacid, M., S. R. Tronick, and S. A. Aaronson. 1978. Isolation and characterization of an endogenous type C RNA virus of mink (MvlLu) cells. J. Virol. 25:129. 14. Ruscetti, S., D. Linemeyer, J. Feild, D. Troxler, and E. Scolnick. 1978. Type-specific radioimmunoassays for the gp70s of mink cell focus-inducing murine leukemia viruses: expression of a cross-reacting antigen in cells infected with the Friend strain of the spleen focus-forming virus. J. Exp. Med. 148:654. 15. Stockert, E., A. B. DeLeo, P. V. O'Donnell, Y. Obata, and L. J. Old. 1978. GIAKSL2~:a new cell surface antigen of the mouse related to the dual tropic MCF class of MuLV detected by naturally occurring antibody. J. Exp. Med. 149:200. 16. O'Donnell, P. V., and E. Stockert. 1976. Induction of Gm antigen and Gross cell surface antigen after infection by ecotropic and xenotropic murine leukemia viruses in vitro.J. Virol. 20:545. 17. Tung, J-S, P. V. O'Donnell, E. Fleissner, and E. A. Boyse. 1978. Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes. J. Exp. Med. 147:1280. 18. Obata, Y., E. Stockert, P. V. O'Donnell, S. Okubo, H. W. Snyder, Jr., and L.J. Old. 1978. GIRADA1): a new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus.J. Exp. Med. 147:1089.
